Nuclear receptors are a family of small molecule and hormone-regulated transcription factors that share conserved DNA-binding (DBD) and ligand-binding domains (LBD). Small pharmacological compounds able to bind to the cleft of the ligand-binding domain could alter its conformation and subsequently modify transcription of target genes. Such ligand agonists and/or antagonists have already been successfully designed for 23 nuclear receptors among the 48 previously identified in the human genome (1-3). Of interest, DAX-1 (NR0B1; dosage-sensitive sex reversal-adrenal hypoplasia congenita critical region on the X chromosome gene 1) has been shown to act as a robust transcriptional repressor, inhibiting genes involved in steroidogenesis through interaction with corepressors and regulating the pluripotency of stem cells (4-8). The human DAX-1 gene encodes a protein whose C terminus is similar to the LBD of nuclear hormone receptors, while its N terminus is composed of three cysteine-rich 70 amino acids with little similarity to known proteins (4, 7). Mutations in Dax-1 have been shown to cause the X-linked form of adrenal hypoplasia congenita (AHC), associated with hypogonadotropic hypogonadism (HHG). AHC-HHG-associated mutations share an altered DAX-1 C-terminal domain (5, 9), resulting in loss of transcriptional repression activity (5, 7, 9). This finding suggests that the impairment of the DAX-1 transcriptional activity is directly linked to the pathogenesis of AHC-HHG. In addition, Dax-1 has also been shown to be highly expressed in pediatric Ewing tumors (10). As a result, the identification of selective inhibitors of Dax-1 will serve as useful tools to elucidate the developmental and tumorigenic roles of Dax-1, and its maintenance of stem cell pluripotency.

Assay Overview:The purpose of this assay is to identify compounds that inhibit the expression of the DAX-1 target gene, CYP11A1. CYP11A1 localizes to the mitochondrial inner membrane and catalyzes the conversion of cholesterol to pregnenolone, the first and rate-limiting step in the synthesis of the steroid hormones. This cell-based assay employs HEK293 cells co-transfected with a full-length DAX-1 expression construct, an SF-1 expression vector (to activate expression of the CYP11A1 reporter) and a CAT reporter harboring the CYP11A1 promoter. As designed, compounds that inhibit DAX-1 activity will reduce the interaction of DAX-1 with corepressors, leading to increased CYP11A1 promoter activity and CAT gene expression, resulting in increased well absorbance. Compounds are tested in triplicate using a 3-point, 1:2 dilution series, starting at a nominal test concentration of 100 uM.Protocol Summary:HEK293 cells were routinely cultured in T-75 flasks containing 15 mL of DMEM (4.5 g/L glucose) medium supplemented with 10% v/v fetal bovine serum and 1% v/v penicillin-streptomycin at 37 C, 5% CO2 and 95% relative humidity (RH). When cells reached about 70% confluency, they were transfected with 1.5 mL of serum-free OptiMEM containing 7.8 ug of the pCYP11A1-CAT reporter plasmid, 3.9 ug of the pSG.SF-1 expression vector and 3.9 ug of the pSV.DAX-1 expression vector, using 31.25 uL of TransIT-293 transfection reagent. In the absence of a pharmacological positive control, DAX-1 inhibition was mimicked by transfecting cells with the empty vector pSG5 instead of pSV.DAX-1. Four hours post transfection, cells were harvested using 3 mL of trypsin-EDTA solution and resuspended at a concentration of 400,000 cells per mL in the wells of 12-well plates (0.5 mL per well), diluted in DMEM supplemented as above. Transfected cells were then incubated at 37 C, 5% CO2 and 95% RH for 16 hours before CAT assay. 0.5 mL of each dilution of the test compounds or DMSO control to reach the appropriate final concentration were also added to the wells. Transfection experiments were performed in duplicate and repeated three times for each tested drug concentration.CAT activity in transfected HEK293 cells was assayed using a colorimetric EIA assay, following the manufacturer's instructions. The relative CAT activity of each test compound was calculated as follows:Relative_Activity = (average absorbance of test compound wells) / (average absorbance of DMSO wells)PubChem Activity Outcome and Score:Compounds that increased relative activity to levels greater than 1.25 at all doses were considered to inhibit DAX1 and are considered active in this assay. Compounds that did not increase relative activity to 1.25 at all doses were considered inactive.The reported PubChem Activity Score has been normalized to 100% observed relative activity at 100 uM.The PubChem Activity Score range for inactive compounds is 100-0. There are no active compounds.List of Reagents:CAT reporter plasmid driven by the CYP11A1 gene promoter (supplied by Assay Provider)DAX-1 expression plasmid (supplied by Assay Provider)SF-1 expression plasmid (supplied by Assay Provider)pSG5 empty plasmid (supplied by Assay Provider)HEK293 cells (ATCC, part CRL-1573)DMEM (Invitrogen, part 11965)FBS (Invitrogen, part 10106-169)Penicillin-Streptomycin 100X Liquid Solution (Invitrogen, part 15140-130)TransIT 293 (Mirus Corporation, part MIR-2700)OptiMEM (Invitrogen, part 31985)Trypsin-EDTA solution (Invitrogen, part 25300-104)CAT ELISA (Roche, part 113637270001)White, clear bottom 96-well plates (Corning, part 3903)

This assay was performed by the assay provider's lab. This assay may have been run as two or more separate campaigns, each campaign testing a unique set of compounds. All data reported were normalized on a per-plate basis. Possible artifacts of this assay can include, but are not limited to: dust or lint located in or on wells of the microtiter plate, and compounds that modulate well absorbance. All test compound concentrations reported above and below are nominal; the specific test concentration(s) for a particular compound may vary based upon the actual sample provided.